Monocytes and macrophages can exhibit non-specific binding of antibodies and fluorophores used in cell surface staining of live cells. BioLegend has now formulated an effective blocking reagent, True-Stain Monocyte Blocker™. It is a non-antibody based blocking solution that has been shown to reduce non-specific monocyte binding due to the fluorophore and does not affect the desirable specific staining of monocytes. Now you can immuno-label monocytes with confidence, fully trusting your true staining results.

There are many fluorophore properties and assay factors that influence the existence or severity of the non-specific binding:

PBMCs vs. whole blood vs. pre-lysed blood

PMA stimulated vs unstimulated cells

Donor-dependent variation

Live vs. fixed cells

Though the specific mechanism of the non-specific binding isn’t known, binding of the cyanine acceptor fluorophores to CD64 has been implicated in the literature. However, in our testing, we have demonstrated that the non-specific binding is not a cyanine fluorophore phenomena alone. PE/Dazzle™ 594, which is not a cyanine-based acceptor fluorophore, also can exhibit non-specific binding to monocytes. Thus the effect appears to be quite selective for any PE, PerCP and APC tandems we have tested. The tandem fluorophores of competitors were not included in our analysis. In our testing, cyanine-based fluorophores like Alexa Fluor® 647 and the cyanine-based acceptors of some of the Brilliant Violet™ tandems do not exhibit non-specific binding to monocytes. To learn more about which fluorophores might require True-Stain Monocyte Blocker™, take a look at the chart to the right.

Fluorophore conjugates recommended for use with True-Stain Monocyte Blocker™

Fluorophore conjugates that likely do not need True-Stain Monocyte Blocker™

Human PBMCs were stained with 5 µl/test of antibody in 100 µl of cells at 1 x 106 cells/ml with (bottom row) or without (top row) True-Stain Monocyte Blocker™.

Blocking Fc Receptors does not prevent non-specific binding of monocytes

The use of the Fc blocker looked identical to samples stained with no blockers at all. If the non-specific binding were mediated by Fc binding, the Fc blocker (Human TruStain FcX™) alone would have fixed the problem. In instances where only the antibody exhibiting non-specific binding (for example, CD3 APC/Cy7, CD56 PE/Cy5 and CD19 PE/Dazzle™ 594) was used to costain cells with CD14 BV421™, the True-Stain Monocyte Blocker™ alone was sufficient to block the non-specific binding. This can be seen in the data above with True-Stain Monocyte Blocker™.

Including many of these tandems in a multicolor panel, as is common for panels starting at 6 colors or more, has a variable effect on the extent of the non-specific binding, as if the presence of many of these tandems competes for the non-specific binding sites and can often diminishes the apparent level of non-specific binding of any single reagent. This effect was seen consistently whether using LWB (lysed whole blood), pre-lysed blood or PBMC.

Table 1.

Description

Clone

CD14 BV421™

HCD14

CD3 APC/Cy7

UCHT1

CD56 PE/Cy5

5.1H11

CD19 PE/Dazzle™ 594

HIB19

CD64 PE/Cy7

10.1

CD4 PerCP/Cy5.5

RPA-T4

HLA-DR Alexa Fluor® 647

L243

Human PBMCs were stained with 5 µl/test of antibody in 100 µl of cells at 1 x 106 cells/ml in a 2-color panel as indicated (top row) or in a 7-color panel as listed in Table 1 (bottom row).